Ligand recognition and G protein coupling of the human itch receptor MRGPRX1

MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception and targeting MRGPRX1 may have potential to treat both chronic itch and pain. Here we report cryo-EM structures of the MRGPRX1-Gi1 and MRGPRX1-Gq trimers in complex with two peptide ligands, BAM8-22 and CNF-Tx2. These structures reveal a shallow orthosteric pocket and its conformational plasticity for sensing multiple different peptidic itch allergens. Distinct from MRGPRX2, MRGPRX1 contains a unique pocket feature at the extracellular ends of TM3 and TM4 to accommodate the peptide C-terminal “RF/RY” motif, which could serve as key mechanisms for peptidic allergen recognition. Below the ligand binding pocket, the G6.48XP6.50F6.51G6.52X(2)F/W6.55 motif is essential for the inward tilting of the upper end of TM6 to induce receptor activation. Moreover, structural features inside the ligand pocket and on the cytoplasmic side of MRGPRX1 are identified as key elements for both Gi and Gq signaling. Collectively, our studies provide structural insights into understanding itch sensation, MRGPRX1 activation, and downstream G protein signaling.

The interaction of peptidic ligands with receptors such as GPCRs is an active area of inquiry for newly developed modeling algorithms. Could the authors perform a simple comparison of experimental structures with those produced by a modeling algorithm such as Alphafold2? Either success or failure of Alphafold2 in predicting structural details would be informative.
On line 52: "GPCRs" should be "GPCR" On line 91 the language used is confusing. Saying that GPCRs are known to couple to TRPA1 is not clear. GPCRs typically couple to G proteins and beta-arrestin. Do these GPCRs couple to TRPA1 in an analogous way or is TRPA1 a downstream response?
On line 224: "was" should be deleted Reviewer #2: Remarks to the Author: The manuscript by Guo et al., reports the cryo-EM structures of MRGPRX1-Gi1 in complex with BAM8-22 or CNF-Tx2 and MRGPRX1-Gq in complex with BAM8-22, revealing a unique shallow ligand binding pocket at the extracellular ends of TM3 and TM4 for peptidic allergen recognition. They also describe the conserved kink motif present in the MRGPRX family for MRGPRX1 activation. In addition, they reveal both the Gi1 and Gq coupling mechanisms of MRGRPX1 and found that TM3 and ICL2 of MRGPRX1 form specific interactions with the bulky end of the α5 helix of Gαq contributed to most of the specific Gαq coupling mechanisms. These observations are nicely verified by their mutagenesis studies. Overall, the manuscript describes elegant and rigorous structural analysis and biochemical experiments. Their maps look like they are good quality. The mechanism that is proposed is reasonable and is based on well-designed experiments that are suggested by the structure. However, before publication could be recommended, a few, mostly minor, issues should be addressed: 1)As far as I know, the structure of Gq-coupled MRGPRX1 with BAM8-22 has been already reported (PDB 8DWC). The authors should compare with their structure and state the differences and similarities.
2)Considering the structure of MRGPRX4-Gq complex has been resolved, it would be better if authors could compare their differences and similarities. 3)Page 3, line 52: "MGRPRX1""MRGPRX1" 4)Page 4, line 91 and 93 "Mrgprc11""MrgprC11" 5)Page 5, line 107, 121 and 123 : "2.9Å""2.98Å" "2.8Å""2.84Å" 6)Page 9, line 218: "is fits" "fits" 7)Page 9, line 224: There is no figure provided to fit the description of the interactions in mode 2. 8)Please check "Cryo-EM data acquisition" again: The pixel size does not fit the pixel size given in Supplementary Table 1, as well as defocus range and total exposure electron. Meanwhile a dose rate of about 7.8 electrons per Å2 per second and total exposure time of 8 s should be the parameters of K2 camera other than K3, the authors need clarify the detailed data collection parameters for each structures. 9)The resolution showed in Supplementary Fig. S2c is 2.85Å which is not consistent with the labeled 2.7Å. 10)In Supplementary Fig. S2d, local resolution of the GPCR part seems unreasonable. 11) Fig. 2c, Supplementary Fig. S2a,b : R20K R20A 12)The clashscore of three structures in Supplementary Table 1 is not consistent with the validation reports. 13)According to the information of 5.3.2 protein sidechains in validation reports, more efforts need to be done to correct outliers. And it would be better to provide formal version of validation reports next time.

Responses to reviewers' comments
We thank the reviewer for their valuable time in reviewing our manuscript and the constructive suggestions that they have provided. We have carefully taken these comments into consideration in preparing a revised version for our manuscript, which has resulted in a more thorough and clear manuscript. Please find below a point-by-point response to the reviewer with our responses in Blue and the reviewers' comments in Red.
Reviewer #1 (Remarks to the Author): Review of "Ligand recognition and G protein coupling of the human itch receptor MRGPRX1" by Guo et al.
In this manuscript the authors present the first structure of full length MRGPRX1, a G-protein coupled receptor involved in itch perception and pathology. Structures of MRGPRX1 in complex with two different ligands (BAM8-22 and CNF-Tx2) reveal the molecular determinants of ligand binding and help to explain the ligand specificity of this receptor relative to other Mas-related GPCRs. Structural characterization of MRGPRX1 in complex with Gi and Gq serve to further elucidate mechanisms of signal transduction.
Reply: We thank the reviewer for his/her positive comments.
This manuscript is a direct follow up to work from a subset of these authors on the structure of MRGPRX2 (reference 13). Related modes of ligand and G protein binding are observed for MRGPRX1 compared to MRGPRX2. The biological and therapeutic implications of characterizing Mas-related GPCR function is high and the structure presented here represents an important and substantial contribution to these efforts. The data presented here are high quality and the conclusions are largely sound (with some technical questions). The work could be suitable for publication in Nature Communications through addressing the comments and questions below.
Reply: We thank the reviewer for his/her positive comments. Reply: We thank the reviewer for his/her helpful suggestions. Previous report has suggested that MRGPRX1 was able to sense endogenous and exogenous peptides sharing a conserved sequence of RF/Y-G or RF/Y-amide near their C-terminal. Here, by comparison of the structures of BAM8-22-MRGPRX1-Gq/Gi and CNF-Tx2-MRGPRX1, as well as the mutational analysis, we were able to identify that a hydrophobic pocket surrounded by Y99 3.29 , L160 4.63 and L240 6.59 of MRGPRX1 played an important role in recognition of both C-terminal Y 21 of BAM8-22 and I 18 of CNF-Tx2. Moreover, the E157 4.60 played central roles in recognition of C-terminal R 17 of CNF-Tx2 and R 20 Y 21 of BAM8-22. In addition to providing structural knowledge for recognition previous proposed C-terminal Rφ motif (φ indicated a hydrophobic residue), we also found that N-terminal to the Rφ motif, the F 15 of CNF-Tx2 is surrounded by large hydrophobic residues of Y82 2.60 , Y99 3.29 , F236 6.55 and H254 7.35 . Similarly, the Y 17 of BAM8-22 is surrounded by large hydrophobic residues of F236 6.55 , F250 7.31 and H254 7.35 of MRGPRX1. Therefore, we proposed that a C-terminal motif of φ B17 (X1-2) R B20 φ B21 in the peptide ligand is more preferred by MRGPRX1 (amino acid position of peptide sequence is named according positions in BAM8-22 peptide). This motif is distinct from our previously identified peptide motif recognized by MRGPRX2, which is φ p9 (X0-1) R/K p10 (X2) φ p13 (X2-3) φ p16 (X3) R/K p20 (Fig. R1a-R1b and Supplementary Fig. 10a-10b in the revised manuscript). We have incorporated these discussions in the "discussion section" of the revised manuscript.
Consistent with these speculations, we have measured the activities of MRGPRX1 toward γ1-MSH, hemoglogbin β-chain, etc, which showed reasonable potency and efficacy, as previously reported (Fig. R1c and Supplementary Fig. 10c in the revised manuscript).
All data were analyzed by two-sided one-way ANOVA with Turkey test. (e) Effects of different CNF-Tx2 mutations on CNF-Tx2 induced Gαi-Gγ dissociation. Data from three independent experiments are presented as the mean ± SEM (n=3). All data were analyzed by two-sided one-way ANOVA with Turkey test.
2) The authors use a MRGPRX1 fused at its N terminus to BRIL for structural studies. Could the authors comment on whether this construct behaves similarly to BRIL-free MRGPRX1 in pharmacological assays? This comparison is important for understanding structural data versus pharmacological data.
Reply: We thank the reviewer for his/her helpful suggestions. To increase receptor expression, thermostabilized cytochrome b562RIL (BRIL) was incorporated at the N-terminus of full-  3) The signaling properties of many ligands and their mutants (as well as receptor mutants) is described with a single pharmacological parameter (pEC50). This seems to leave out important information about maximal responses (efficacy). Addition of this information would be helpful for evaluating the impact of mutations on signaling.
Reply: We thank the reviewer for his/her helpful suggestions. We have added the Emax data according to this reviewer's suggestion in the revised manuscript in Fig are measured as mean ± SEM (n=3). All data were determined by two-sided one-way ANOVA with Tukey test. ***, P < 0.001, n.s., no significant difference. Fig. 9 Binding of CNF-Tx2 to MRGPRX1.  P1: Gαi-Gγ dissociation assay data， P2: Gαq-Gγ dissociation assay data， β>0， Gi biased; β<0，
Therefore, as shown in the Fig. R2, chloroquine (CQ) is a Gq bias ligand, whereas CNF-Tx2 showed Gi bias when we compared Gq activation over Gi using BAM8-22 as a reference. it seems that deleting the C terminal VRI motif would allow assessment of the importance of these residues for receptor activation. Is there a reason not to test this directly?
Reply: We thank the reviewer for his/her helpful suggestions. As suggested by the reviewer, we have synthesized the truncated CNF-Tx2 peptide (deleting the C terminal VRI motif) and found that the truncation almost abolished the activity of MRGPRX1 compared to the CNF-Tx2 wide-type peptide (Fig. R4, also shown in Fig. 3d and Supplementary Fig. 9a-9b in the revised manuscript). Fig. 3d and Supplementary Fig. S9a-S9b).

Fig. R4 (also shown in
Effects of the wide-type and truncated CNF-Tx2 peptides induced Gαi-Gγ dissociation in MRGPRX1 overexpressing HEK293 cells. The curve data from three independent measurements are measured as mean ± SEM (n=3). ND, not detectable due to low signal. These residues may directly interact with MRGPRX1 but we didn't observe these interactions.
In addition, the binding energy is the sum of entropy and enthalpy. Whereas interactions contributed to the enthalpy, the entropy is mostly dependent on conformations of ligands and receptors. We speculated that several residues of BAM8-22 played important roles in entropy changes when the BAM8-22 binds to MRGPRX1, therefore contributing to the binding energy of the peptide without direct interactions with the MRGPRX1. We have incorporated these discussions in the "Binding of BAM8-22 to MRGPRX1" of the revised manuscript.
7) The interaction of peptidic ligands with receptors such as GPCRs is an active area of inquiry for newly developed modeling algorithms. Could the authors perform a simple comparison of experimental structures with those produced by a modeling algorithm such as Alphafold2?
Either success or failure of Alphafold2 in predicting structural details would be informative.
Reply: We thank the reviewer for his/her helpful suggestions. As far as we know, Alphafold2 can currently only be used to predict protein structure but not protein-protein interactions. A recent article that has not yet been officially published mentioned that AlphaFold version 2.3 was explicitly trained to model protein-protein interactions, but this program requires large amounts of physical space, and we are still working on it. In addition, using the ZDOCK program, which is commonly used to predict the binding patterns of peptide ligands to receptors, we found that the top two predicted results were quite different from the binding patterns of the ligands in our resolved structures (Fig. R5), so we speculated the predictions at the current stage were still not comparable to experimental data. 8) On line 52: "GPCRs" should be "GPCR" Reply: We thank the reviewer for his/her helpful suggestions, we have replaced the "GPCRs" with "GPCR" in our revised manuscript: Line 45 -'MRGPRX1, a Mas-related GPCR (MRGPR), is a key receptor for itch perception, and targeting MRGPRX1 may have the potential to treat both chronic itch and pain.' 9) On line 91 the language used is confusing. Saying that GPCRs are known to couple to TRPA1 is not clear. GPCRs typically couple to G proteins and beta-arrestin. Do these GPCRs couple to TRPA1 in an analogous way or is TRPA1 a downstream response?
Reply: We thank the reviewer for his/her helpful suggestions. We have replaced the term of 'couple to' with 'functionally link to' and 'link to' according to this reviewer's suggestion in the revised manuscript, which includes the following places: Line 82 -'The functional homologs of MRGPRX1 in mice, MrgprA3 and MrgprC11, are known to functionally link to TRPA1, which is essential for itch sensation.' Line 83-'These two receptors are functionally link to TRPA1 through different mechanisms.
Whereas Mrgprc11 connected to TRPA1 through Gq-PLC signaling, MrgprA3 was found to link to TRPA1 through Gβγ. Using dental afferents of human samples, MRGPRX1 was shown to sensitize TRPA1 and instigate membrane depolarization 10, 11 .' 10) On line 224: "was" should be deleted Reply: We thank the reviewer for his/her helpful suggestions, we have deleted the word 'was' on line 214 in our revised manuscript.
Reviewer #2 (Remarks to the Author): The Overall, the manuscript describes elegant and rigorous structural analysis and biochemical experiments. Their maps look like they are good quality. The mechanism that is proposed is reasonable and is based on well-designed experiments that are suggested by the structure.
However, before publication could be recommended, a few, mostly minor, issues should be addressed: Reply: We thank the reviewer for his/her positive comments.   6) Page 9, line 218: "is fits" "fits" Reply: We thank the reviewer for his/her helpful suggestions. We have revised it in our revised manuscript as follows: Line 206："Compared with mode 2 the CNF-Tx2 in mode 1 fits better with EM density. We then performed a molecular dynamics simulation by including side chain atoms that were not defined by EM density and the result indicated that model 1 was more stable (Fig. 3e).'' 7) Page 9, line 224: There is no figure provided to fit the description of the interactions in mode 2.
Reply: We thank the reviewer for his/her helpful suggestions. We have added Fig. R8 (also shown in Supplementary Fig. 9c) to fit the description of the interactions in mode 2: "Compared with mode 1, the CNF-Tx2 in mode 2 was lost specific interactions with E157 4.60 and D177 5.36 and formed new contact with F239 6.58 ."

Cryo-EM data acquisition
The For the BAM8-22-MRGPRX1-Gq complex dataset, 5,601 movies were collected on a Titan Krios equipped with a Gatan K3 direct electron detection device at 300 kV with a magnification of 81,000, corresponding to a pixel size 1.04 Å. We collected a total of 36 frames accumulating to a total dose of 50 e − /Å2 over 2.5 s exposure on each TIF format movie.
For the CNF-Tx2-MRGPRX1-Gi1 complexes, 3,085 movies were collected on a Titan Krios equipped with a Gatan K2 direct electron detection device at 300 kV with a magnification of 130,000, corresponding to a pixel size 1.08 Å. The total exposure time was 8 s, resulting in an accumulated dose of 50 electrons per Å2 and a total of 32 frames per movie.
For the BAM8-22-MRGPRX1-Gi1 complexes, all 5,540 movies were collected on a Titan Krios equipped with a Gatan K3 direct electron detection device at 300 kV with a magnification of 130,000, corresponding to a pixel size 0.89 Å. The total exposure time was 3 s, resulting in an accumulated dose of 60 electrons per Å2 and a total of 32 frames per movie. Reply: We thank the reviewer for his/her helpful suggestions. We have checked the single particle reconstruction of the BAM8-22-MRGPRX1-Gq complex, and revised the resolution to 2.9 Å in Supplementary Fig. 2c: 10) In Supplementary Fig. S2d, local resolution of the GPCR part seems unreasonable.

Supplementary
Reply: We thank the reviewer for his/her very helpful suggestions. We have replaced the new 3D density map colored according to the local resolution (Å) of the BAM8-22-MRGPRX1-Gq trimer complex as shown above. Fig. 2c, Supplementary Fig. S2a There are a few small follow up questions for which I request further responses.

11)
• For the new Emax data (Comment 3) it was striking that almost all mutations cause a reduction in Emax relative to the wild type receptor. It can be tricky to interpret Emax data as differences in either receptor functionality or receptor localization can lead to variation in Emax. Could the authors provide a discussion on why there seem to be such dramatic effects on Emax levels? Are mutations known to affect receptor stability or trafficking?
• The new analysis of ligand bias (comment 4) is interesting but it seems only to be included as a figure for review. I think this should be included as a supporting figure unless there is a compelling reason to exclude it. • There has been extensive analysis of protein-protein interactions (comment 7) applying modeling from Alphafold2 in Alphafold multimer (PMID: 35900023). Web tools such as Colabfold (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) make this straightforward. This approach has been applied to other GPCR/ligand pairs (https://pubmed.ncbi.nlm.nih.gov/37092865/). I think a comparison of experimental data with Alphafold2-based modeling results would be of wide interest.
Reviewer #2: Remarks to the Author: The authors have made an effort to address my concerns. However I still have two minor questions that should be addressed before supporting publication of the revised manuscript.
1.The local resolution map showed in Supplementary Fig 2d still

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): The authors have provided new data and discussion to effectively address my previous comments. There are a few small follow up questions for which I request further responses.
Reply: We thank the reviewer for his/her positive comments.
1. For the new Emax data (Comment 3) it was striking that almost all mutations cause a reduction in Emax relative to the wild type receptor. It can be tricky to interpret Emax data as differences in either receptor functionality or receptor localization can lead to variation in Emax.
Could the authors provide a discussion on why there seem to be such dramatic effects on Emax levels? Are mutations known to affect receptor stability or trafficking?
Reply: We thank the reviewer for his/her helpful suggestions. We have re-examined the expression level of wild type and mutants on the cell membrane and the Emax values were updated after revision of the transfecting plasmid amounts to enable similar plasma membrane expression of the wild type or mutant MRGPRX1 receptors. As results, several mutants were not responsive in response to BAM-22 or CNF stimulation. Approximately 20%~30% mutants showed significant effects on Emax, whereas more than 60% of mutants showed no significant change for Emax. The EC50 didn't show significant changes compared with previous version.
We have included the new data in the revised manuscript in Fig. 2d, Supplementary Fig. 8c, Supplementary Fig. 10d-10f, Supplementary Fig. 12c-12d and Supplementary Fig. 12g-12h as follows: The curve data from three independent measurements are measured as mean ± SEM (n=3). All data were determined by two-sided one-way ANOVA with Tukey test. ***, P < 0.001, n.s., no significant difference.     Fig. 10a-10c). We therefore speculated that the experimental data is still needed for analyzing the interaction between peptide ligand and their corresponding receptors."